Intracellular delivery of biomolecules to cells comprising a cell wall

ABSTRACT

The present disclosure pertains to methods for delivering a compound into a cell comprising a cell wall, including passing a cell suspension through a constriction, wherein said constriction deforms the cell comprising a cell wall, thereby causing a perturbation of the cell such that the compound enters the cell, wherein said cell suspension is contacted with the compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/757,316, which is a National Stage of International Application No. PCT/US2016/050288, filed on Sep. 2, 2016, which claims the priority benefit of U.S. Provisional Application No. 62/214,821, filed on Sep. 4, 2015, and U.S. Provisional Application No. 62/365,177, filed on Jul. 21, 2016, all of which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present disclosure relates generally to methods for delivering a compound into a cell comprising a cell wall by passing a cell suspension through a constriction.

BACKGROUND

Intracellular delivery is a central step in the research and development of engineered organisms. Existing technologies aimed at intracellular delivery of molecules rely on electrical fields, nanoparticles, or pore-forming chemicals. However, these methods suffer from numerous complications, including non-specific molecule delivery, modification or damage to the payload molecules, high cell death, low throughput, and/or difficult implementation. In addition, these intracellular delivery methods are not effective at delivering molecules to cells with cell walls, such as plant, yeast, fungal, algal, and prokaryotic cells. Thus, there is an unmet need for intracellular delivery techniques that are highly effective at delivering a range of molecules to a variety of cell types. In addition, techniques that allow for rapid, high throughput intracellular delivery can be applied more effectively to large scale clinical, manufacturing, and drug screening applications. References that describe methods of using channels to deliver compounds to cells include WO2013059343 and WO2015023982.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

Certain aspects of the present disclosure include a method for delivering a compound into a cell comprising a cell wall, the method comprising passing a cell suspension through a constriction, wherein said constriction deforms the cell comprising a cell wall, thereby causing a perturbation of the cell such that the compound enters the cell, wherein said cell suspension is contacted with the compound.

Other aspects of the present disclosure include a method for delivering a compound into a cell modified to remove (such as digest or disrupt) all or part of a cell wall, the method comprising passing a cell suspension through a constriction, wherein said constriction deforms the cell modified to remove all or part of a cell wall, thereby causing a perturbation of the cell such that the compound enters the cell, wherein said cell suspension is contacted with the compound

In some embodiments that can be combined with the previous embodiments, the constriction is contained within a microfluidic channel. In some embodiments, the constriction is a pore or contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the constriction size is a function of the cell diameter. In some embodiments, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.

In some embodiments that can be combined with the previous embodiments, the cell suspension comprises a mixed cell population. In some embodiments, the cell suspension comprises a purified cell population. In some embodiments, the cell comprising a cell wall is a plant, yeast, fungal, algal, or prokaryotic cell. In some embodiments, the plant cell is a crop, model, ornamental, vegetable, leguminous, conifer, or grass plant cell. In some embodiments, the yeast cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain cell. In some embodiments, the fungal cell is an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma strain cell. In some embodiments, the prokaryotic cell is a Bacillus coagulans, Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Carboxydocella sp., Clostridium perfringens, Clostridium septicum, Clostridium tetani, Corynebacterium glutamicum, Enterobacteriaceae, Enterococcus faecalis, Erwinia chrysanthemi, Faecalibacterium prausnitzii, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sp., Pediococcus acidilactici, Peptococcus sp., Peptostreptococcus sp., Propionibacterium freudenreichii, Proteus mirabilis, Pseudomonas aeruginosa, Rhodopseudomonas capsulata, Salmonella enteritidis, Staphylococcus aureus, Streptococcus faecium, Streptococcus lactis, Streptococcus salivarius, Streptococcus thermophilus, Vibrio furnissii, Caldicellulosiruptor saccharolyticus, Xanthomonas campestris, cyanobacteria, Escherichia, Enterobacter, Azotobacter, Envinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, or Paracoccus cell. In some embodiments, the algal cell is a Chlorophyceae, Bacillariophyceae, Eustigmatophyceae, or Chrysophyceae cell.

In some embodiments that can be combined with the previous embodiments, the compound comprises a nucleic acid. In some embodiments, the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplifying mRNA. In some embodiments, the compound comprises a peptide nucleic acid. In some embodiments, the compound comprises a transposon. In some embodiments, the compound is a plasmid. In some embodiments, the compound comprises a plastid. In some embodiments, the compound comprises a protein-nucleic acid complex. In some embodiments, the compound comprises a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the compound comprises nucleic acid encoding for a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the compound comprises a lipid-nucleic acid complex. In some embodiments, the compound comprises a cation-nucleic acid complex. In some embodiments, the compound comprises a protein or peptide. In some embodiments, the compound comprises a TALEN protein, Zinc finger nuclease, mega nuclease, CRE recombinase, FLP recombinase, R recombinase, integrase, or transposase. In some embodiments, the compound comprises a histone acetyltransferase, deacetylase, methyltransferase, or demethylase. In some embodiments, the compound is an antibody. In some embodiments, the compound is a transcription factor. In some embodiments, the compound is a small molecule. In some embodiments, the compound is a nanoparticle. In some embodiments, the compound is a liposome. In some embodiments, the compound is a fluorescently tagged molecule. In some embodiments, said cell suspension is contacted with the compound before, concurrently, or after passing through the constriction.

In some embodiments that can be combined with the previous embodiments, the cell is modified to remove all or part of the cell wall. In some embodiments, the cell suspension is treated with an enzyme to digest all or part of the cell wall prior to passing the cell suspension through the constriction. In some embodiments, the enzyme is one or more of cellulase, pectinase, xylanase, lysozyme, zymolyase, or chitinase. In some embodiments, the cell suspension is treated with heat or ultrasound to disrupt all or part of the cell wall prior to passing through the constriction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of flow cytometry to identify live cells and cells incorporating a fluorescent dye following constriction-mediated delivery in S. cerevisiae cells.

FIG. 2 shows the results of selection on YPD+NTC plates for S. cerevisiae cells following constriction-mediated delivery of a plasmid conferring NTC resistance. NTC-resistant colonies indicated by arrows.

DETAILED DESCRIPTION

Certain aspects of the present disclosure relate to methods for delivering a compound into a cell comprising a cell wall or modified to remove all or part of a cell wall, the methods including passing a cell suspension through a constriction, wherein said constriction deforms the cell comprising a cell wall or modified to remove all or part of a cell wall, thereby causing a perturbation of the cell such that the compound enters the cell, wherein said cell suspension is contacted with the compound. In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, the constriction is a pore or contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane.

I. GENERAL TECHNIQUES

The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4^(th) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols in Molecular Biology (DOI: 10.1002/0471142727); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds., 1995); Antibodies, A Laboratory Manual (E. A. Greenfield, eds., 2013); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R. I. Freshney, 6^(th) ed., J. Wiley and Sons, 2010); Oligonucleotides and Analogues (F. Eckstein, ed., 1992); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Handbook (J. E. Celis, ed., Academic Press, 2005); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, Springer, 2013); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (DOI: 10.1002/0471142735); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Janeway's Immunobiology (K. Murphy and C. Weaver, Garland Science, 2016); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); Making and Using Antibodies: A Practical Handbook (G. C. Howard and M. R. Kaser, eds., CRC Press, 2013); The Antibodies Vol. 1-7 (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995-2007); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 2011).

II. DEFINITIONS

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise.

It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.

For all compositions described herein, and all methods using a composition described herein, the compositions can either comprise the listed components or steps, or can “consist essentially of” the listed components or steps. When a composition is described as “consisting essentially of” the listed components, the composition contains the components listed, and may contain other components which do not substantially affect the methods disclosed, but do not contain any other components which substantially affect the methods disclosed other than those components expressly listed; or, if the composition does contain extra components other than those listed which substantially affect the methods disclosed, the composition does not contain a sufficient concentration or amount of the extra components to substantially affect the methods disclosed. When a method is described as “consisting essentially of” the listed steps, the method contains the steps listed, and may contain other steps that do not substantially affect the methods disclosed, but the method does not contain any other steps which substantially affect the methods disclosed other than those steps expressly listed. As a non-limiting specific example, when a composition is described as ‘consisting essentially of’ a component, the composition may additionally contain any amount of pharmaceutically acceptable carriers, vehicles, or diluents and other such components which do not substantially affect the methods disclosed.

The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

The term “cell wall” as used herein refers to an outer layer that surrounds certain cell types, such as plants, yeast, fungi, algae, and some prokaryotes. In some examples the term refers to a tough or rigid layer that surrounds the plasma membrane and provides support to the cell. In some examples, the cell wall may be composed of a number of materials, including without limitation, cellulose, hemicellulose, pectin, peptidoglycan, glucosamine, chitin, glycoproteins, or polysaccharides.

The term “constriction” as used herein refers to a narrowed passageway. In some examples, the constriction is contained within a microfluidic channel. In other examples, the constriction is a pore or contained within a pore. In some examples where the constriction is a pore, the pore is contained in a surface.

The term “pore” as used herein refers to an opening, including without limitation, a hole, tear, cavity, aperture, break, gap, or perforation within a material. In some examples, (where indicated) the term refers to a pore within a surface of the present disclosure. In other examples, (where indicated) a pore can refer to a pore in a cell wall and/or cell membrane.

The term “membrane” as used herein refers to a selective barrier or sheet containing pores. The term includes a pliable sheetlike structure that acts as a boundary or lining. In some examples, the term refers to a surface or filter containing pores. This term is distinct from the term “cell membrane”.

The term “filter” as used herein refers to a porous article that allows selective passage through the pores. In some examples the term refers to a surface or membrane containing pores.

The term “heterogeneous” as used herein refers to something which is mixed or not uniform in structure or composition. In some examples the term refers to pores having varied sizes, shapes or distributions within a given surface.

The term “homogeneous” as used herein refers to something which is consistent or uniform in structure or composition throughout. In some examples the term refers to pores having consistent sizes, shapes, or distribution within a given surface.

The term “heterologous” as used herein refers to a molecule which is derived from a different organism. In some examples the term refers to a nucleic acid or protein which is not normally found or expressed within the given organism.

The term “homologous” as used herein refers to a molecule which is derived from the same organism. In some examples the term refers to a nucleic acid or protein which is normally found or expressed within the given organism.

The term “polynucleotide” or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

For any of the structural and functional characteristics described herein, methods of determining these characteristics are known in the art.

III. CELL SUSPENSIONS

In certain aspects, the present disclosure relates to passing a cell suspension through a constriction. In some embodiments, the cell suspension comprises a cell comprising a cell wall. In some embodiments, the cell suspension comprises a cell modified to remove all or part of a cell wall. In some embodiments, the cell comprising a cell wall or modified to remove all or part of a cell wall is a plant, yeast, fungal, algal, or prokaryotic cell. The cell suspension may be a mixed or purified population of cells. In some embodiments, the cell comprising a cell wall or modified to remove all or part of a cell wall is a eukaryotic cell.

In some embodiments, the cell comprising a cell wall or modified to remove all or part of a cell wall is a plant cell. In some embodiments, the plant cell is a crop, model, ornamental, vegetable, leguminous, conifer, or grass plant cell. Suitable plants include both monocotyledonous (monocot) plants and dicotyledonous (dicot) plants. In some embodiments, the plant cell is a plant cell line cell. In some embodiments, the plant cell is a recombinant plant cell. In some embodiments, the plant cell is a cultured plant cell.

Examples of crop plants, model plants, and vegetables include, without limitation, Arabidopsis, corn, rice, alfalfa, sunflower, canola, cotton, sorghum, wheat, tobacco, oat, barley, lemna, tomato, lettuce.

Examples of ornamental plants include, without limitation, azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbiapulcherrima), and chrysanthemum.

Examples of leguminous plants include, without limitation, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, green beans (Phaseolus vulgaris), peas (Lathyrus spp.), lima beans (Phaseolus limensis), fava bean, lentils, chickpea, peanuts (Arachis sp.), crown vetch (Vicia sp.), hairy vetch, adzuki bean, lupine (Lupinus sp.), trifolium, common bean (Phaseolus sp.), field bean (Pisum sp.), clover (Melilotus sp.), Lotus, trefoil, lens, and false indigo.

Examples of conifer plants include, without limitation, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), Monterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii), Western hemlock (Isuga canadensis), Sitka spruce (Picea glauca), redwood (Sequoia sempervirens), silver fir (Abies amabilis), balsam fir (Abies balsamea), Western red cedar (Thuja plicata), and Alaska yellow-cedar (Chamaecyparis nootkatensis).

Examples of grass plants include, without limitation, alfalfa (Medicago sp.), orchard grass, tall fescue, perennial ryegrass, creeping bent grass, and redtop.

In some embodiments, the cell comprising a cell wall or modified to remove all or part of a cell wall is a yeast cell. In some embodiments, the yeast cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and Fungi Imperfecti (Blastomycetes) yeast. In some embodiments, the yeast cell is a yeast cell line cell. In some embodiments, the yeast cell is a recombinant yeast cell. In some embodiments, the yeast cell is a cultured yeast cell.

In some embodiments, the yeast cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces pombe, or Saccharomyces oviformis strain cell. In some embodiments, the yeast cell is a Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces marxiamus, Pichia stipitis, Candida shehatae, or Candida tropicalis cell. In other embodiments, the yeast cell is a Yarrowia lipolytica, Brettanomyces custersii, or Zygosaccharomyces roux cell.

In some embodiments, the cell comprising a cell wall or modified to remove all or part of a cell wall is a fungal cell. In some embodiments, the fungal cell is an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma strain cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi. In some embodiments, the fungal cell is a fungal cell line cell. In some embodiments, the fungal cell is a recombinant fungal cell. In some embodiments, the fungal cell is a cultured fungal cell.

In embodiments, the fungal cell is a filamentous fungal strain cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota. In some embodiments, the filamentous fungal cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae strain cell. In other embodiments, the filamentous fungal cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum strain cell. In other embodiments, the filamentous fungal cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Scytalidium thermophilum, Sporotrichum thermophile, or Thielavia terrestris strain cell. In some embodiments, the filamentous fungal cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain cell.

In some embodiments, the cell comprising a cell wall or modified to remove all or part of a cell wall is a prokaryotic cell. In some embodiments, the prokaryotic cell is a prokaryotic cell line cell. In some embodiments, the prokaryotic cell is a recombinant prokaryotic cell. In some embodiments, the prokaryotic cell is a cultured prokaryotic cell. In some embodiments, the prokaryotic cell is a gram-positive bacteria cell. Gram-positive bacteria have a cell wall comprising a thick peptidoglycan layer. In some embodiments, the prokaryotic cell is a gram-negative bacteria cell. Gram-negative bacterial have a cell wall comprising a thin peptidoglycan layer between an inner cytoplasmic cell membrane and an outer membrane. In some embodiments, the prokaryotic cell is a Bacillus coagulans, Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oxalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Carboxydocella sp., Clostridium perfringens, Clostridium septicum, Clostridium tetani, Corynebacterium glutamicum, Enterobacteriaceae, Enterococcus faecalis, Erwinia chrysanthemi, Faecalibacterium prausnitzii, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sp., Pediococcus acidilactici, Peptococcus sp., Peptostreptococcus sp., Propionibacterium freudenreichii, Proteus mirabilis, Pseudomonas aeruginosa, Rhodopseudomonas capsulata, Salmonella enteritidis, Staphylococcus aureus, Streptococcus faecium, Streptococcus lactis, Streptococcus salivarius, Streptococcus thermophilus, Vibrio furnissii, Caldicellulosiruptor saccharolyticus, Xanthomonas campestris, cyanobacteria, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, or Paracoccus cell. In some embodiments, the prokaryotic cell is an E. coli, Bacillus subtilis, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum (Moorella thermoacetica), Thermoanaerobacterium saccharolyticum, Carboxydocella sp., Corynebacterium glutamicum, Enterobacteriaceae, Erwinia chrysanthemi, Lactobacillus sp., Pediococcus acidilactici, Rhodopseudomonas capsulata, Streptococcus lactis, Vibrio furnissii, Caldicellulosiruptor saccharolyticus, or Xanthomonas campestris cell.

In some embodiments, the cell comprising a cell wall or modified to remove all or part of a cell wall is an algal cell. In some embodiments, the algal cell is a Chlorophyceae, Bacillariophyceae, Eustigmatophyceae, or Chrysophyceae cell. In some embodiments, the algal cell is an algal cell line cell. In some embodiments, the algal cell is a recombinant algal cell. In some embodiments, the algal cell is a cultured algal cell. In some embodiments, the algal cell is a Chlamydomonas reinhardtii cell. In some embodiments, the algal cell is a Dunaliella cell. In some embodiments, the algal cell is a Chlorella cell. In some embodiments, the algal cell is a Spirulina cell.

The composition of the cell suspension (e.g., osmolarity, salt concentration, ionic strength, protein content, cell concentration, pH, redox potential, etc.) can impact delivery of the compound. In some embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises cell culture medium, PBS, salts, sugars, growth factors, plant products, fungal products, yeast extract, bulking materials, surfactants, lubricants, vitamins, or proteins. Additionally, solution buffer can include one or more lubricants (pluronics or other surfactants) that can be designed to reduce or eliminate clogging of the constriction and improve cell viability.

IV. MICROFLUIDIC CHANNELS

In certain aspects, the present disclosure relates to methods for delivering a compound into a cell comprising a cell wall or modified to remove all or part of a cell wall including the steps of passing a cell suspension through a constriction, wherein the constriction deforms the cell comprising a cell wall or modified to remove all or part of a cell wall, causing a perturbation of the cell, and contacting the cell suspension with the compound; e.g., before, during or after the cells in the suspension pass through the constriction. In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, multiple constrictions can be placed in parallel and/or in series within the microfluidic channel.

In some embodiments, the microfluidic channel includes a lumen and is configured such that a cell suspended in a buffer can pass through, wherein the microfluidic channel includes a constriction. The microfluidic channel can be made of any one of a number of materials, including silicon, metal (e.g., stainless steel), plastic (e.g., polystyrene), ceramics, glass, crystalline substrates, amorphous substrates, or polymers (e.g., Poly-methyl methacrylate (PMMA), PDMS, Cyclic Olefin Copolymer (COC), etc.). Fabrication of the microfluidic channel can be performed by any method known in the art, including dry etching, wet etching, photolithography, injection molding, laser ablation, or SU-8 masks.

In some embodiments, the constriction within the microfluidic channel includes an entrance portion, a centerpoint, and an exit portion. In some embodiments, the length, depth, and width of the constriction within the microfluidic channel can vary. In some embodiments, the diameter of the constriction within the microfluidic channel is a function of the diameter of the cell comprising a cell wall or modified to remove all or part of a cell wall. In some embodiments, the diameter of the constriction within the microfluidic channel is about 20% to about 99% of the diameter of the cell. In some embodiments, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. The cross-section of the channel, the entrance portion, the centerpoint, and the exit portion can also vary. For example, the cross-sections can be circular, elliptical, an elongated slit, square, hexagonal, or triangular in shape. The entrance portion defines a constriction angle, wherein the constriction angle is optimized to reduce clogging of the channel. The angle of the exit portion can vary as well. For example, the angle of the exit portion is configured to reduce the likelihood of turbulence that can result in non-laminar flow. In some embodiments, the walls of the entrance portion and/or the exit portion are linear. In other embodiments, the walls of the entrance portion and/or the exit portion are curved.

In some embodiments, the cross-sectional area of the pore is a function of the cross-sectional area of the cell. In some embodiments, the two-dimensional shape of the pore is circular, elliptical, square, rectangular, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal and the cross-sectional area of the pore is a function of the cross-sectional area of the cell. In some embodiments, the pore cross-sectional area is at least about 1 μm², 4 μm², 9 μm², 16 μm², 25 μm², 50 μm², 100 μm², 150 μm², 200 μm², 250 μm²500 μm² or 1000 μm². In some embodiments, the pores are heterogeneous in cross-sectional area or homogeneous in cross-sectional area across a given surface. In some embodiments, the heterogeneous pore cross-sectional area varies from 10-20% or any percentage or range of percentages therebetween. In some embodiments, the pore deforms the cell to about any one of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the cross-sectional area of the cell or any value therebetween. In some embodiments, the size of the cell is the size of the cell in suspension.

V. SURFACE HAVING PORES

In certain aspects, the present disclosure relates to methods for delivering a compound into a cell comprising a cell wall or modified to remove all or part of a cell wall comprising the steps of passing a cell suspension through a constriction, wherein the constriction deforms the cell comprising a cell wall or modified to remove all or part of a cell wall, causing a perturbation of the cell, and contacting the cell suspension with the compound; e.g., before, during or after the cells in the suspension pass through the constriction, wherein the compound enters the cell. In some embodiments, the constriction is a pore or contained within a pore. In some embodiments, the pore is contained in a surface.

The surfaces as disclosed herein can be made of any one of a number of materials and take any one of a number of forms. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface is a sponge or sponge-like matrix. In some embodiments, the surface is a matrix.

In some embodiments the surface is a tortuous path surface. In some embodiments, the tortuous path surface comprises cellulose acetate. In some embodiments, the surface comprises a material selected from, without limitation, synthetic or natural polymers, polycarbonate, silicon, glass, metal, alloy, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, Polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluorethylene, mixed cellulose ester, porcelain, and ceramic.

The surface disclosed herein can have any shape known in the art; e.g. a 3-dimensional shape. The 2-dimensional shape of the surface can be, without limitation, circular, elliptical, round, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal. In some embodiments, the surface is round in shape. In some embodiments, the surface 3-dimensional shape is cylindrical, conical, or cuboidal.

The surface can have various cross-sectional widths and thicknesses. In some embodiments, the surface cross-sectional width is between about 1 mm and about 1 m or any cross-sectional width or range of cross-sectional widths therebetween. In some embodiments, the surface has a defined thickness. In some embodiments, the surface thickness is uniform. In some embodiments, the surface thickness is variable. For example, in some embodiments, portions of the surface are thicker or thinner than other portions of the surface. In some embodiments, the surface thickness varies by about 1% to about 90% or any percentage or range of percentages therebetween. In some embodiments, the surface is between about 0.01 μm to about 0.02 μm, about 0.02 μm to about 0.03 μm, about 0.03 μm to about 0.04 μm, about 0.04 μm to about 0.05 μm, about 0.05 μm to about 0.06 μm, about 0.06 μm to about 0.07 μm, about 0.07 μm to about 0.08 μm, about 0.08 μm to about 0.09 μm, about 0.09 μm to about 0.1 μm, about 0.1 μm to about 0.2 μm, about 0.2 μm to about 0.3 μm, about 0.3 μm to about 0.4 μm, about 0.4 μm to about 0.5 μm, about 0.5 μm to about 0.6 μm, about 0.6 μm to about 0.7 μm, about 0.7 μm to about 0.8 μm, about 0.8 μm to about 0.9 μm, about 0.9 μm to about 1 μm, about 1 μm to about 2 μm, about 2 μm to about 3 μm, about 3 μm to about 4 μm, about 4 μm to about 5 μm, about 5 μm to about 6 μm, about 6 μm to about 7 μm, about 7 μm to about 8 μm, about 8 μm to about 9 μm, about 9 μm to about 10 μm, about 10 μm to about 20 μm, about 20 μm to about 30 μm, about 30 μm to about 40 μm, about 40 μm to about 50 μm, about 50 μm to about 60 μm, about 60 μm to about 70 μm, about 70 μm to about 80 μm, about 80 μm to about 90 μm, about 90 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 300 μm, about 300 μm to about 400 μm, about 400 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm to about 700 μm, about 700 μm to about 800 μm, about 800 μm to about 900 μm, about 900 μm to about 1 mm, about 1 mm to about 2 mm, about 2 mm to about 3 mm, about 3 mm to about 4 mm, about 4 mm to about 5 mm thick or any thickness or range of thicknesses therebetween. In some embodiments, the surface is between about 0.01 μm to about 5 mm thick or any thickness or range of thicknesses therebetween.

In some embodiments, the constriction is a pore or contained within a pore. The cross-sectional width of the pores is related to the type of cell to be treated. In some embodiments, the pore size is a function of the diameter of the cell to be treated. In some embodiments, the pore size is such that a cell is perturbed upon passing through the pore. In some embodiments, the pore size is less than the diameter of the cell. In some embodiments, the pore size is about 20% to about 99% of the diameter of the cell. In some embodiments, the pore size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. Optimal pore size can vary based upon the application and/or cell type.

The entrances and exits of the pore passage may have a variety of angles. The pore angle can be selected to minimize clogging of the pore while cells are passing through. In some embodiments, the flow rate through the surface is between about 0.001 mL/cm²/sec to about 100 L/cm²/sec or any rate or range of rates therebetween. For example, the angle of the entrance or exit portion can be between about 0 and about 90 degrees. In some embodiments, the pores have identical entrance and exit angles. In some embodiments, the pores have different entrance and exit angles. In some embodiments, the pore edge is smooth, e.g. rounded or curved. A smooth pore edge has a continuous, flat, and even surface without bumps, ridges, or uneven parts. In some embodiments, the pore edge is sharp. A sharp pore edge has a thin edge that is pointed or at an acute angle. In some embodiments, the pore passage is straight. A straight pore passage does not contain curves, bends, angles, or other irregularities. In some embodiments, the pore passage is curved. A curved pore passage is bent or deviates from a straight line. In some embodiments, the pore passage has multiple curves, e.g. about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more curves.

The pores can have any shape known in the art, including a 2-dimensional or 3-dimensional shape. The pore shape (e.g., the cross-sectional shape) can be, without limitation, circular, elliptical, round, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross-section of the pore is round in shape. In some embodiments, the 3-dimensional shape of the pore is cylindrical or conical. In some embodiments, the pore has a fluted entrance and exit shape. In some embodiments, the pore shape is homogenous (i.e. consistent or regular) among pores within a given surface. In some embodiments, the pore shape is heterogeneous (i.e. mixed or varied) among pores within a given surface.

The terms “pore size” and “pore cross-sectional width” are used interchangeably, and as used herein refer to the smallest cross-sectional width across the pore. In some embodiments, the pore is circular or roughly circular and the pore size or pore cross sectional width. In some embodiments, the pore is polygonal in shape (e.g., square, rectangular, pentagonal, hexagonal, etc.) and the pore size or pore cross sectional width is the smallest width of the polygon. One skilled in the art would understand that a triangular pore may not have a width, but rather, is described in terms of bases and heights. In some embodiments, the pore size or pore cross sectional width of a triangular pore is the smallest height of the triangle (smallest distance between a base and its opposite angle).

The surfaces described herein can have a range of total pore numbers. In some embodiments, the pores encompass about 10% to about 80% of the total surface area. In some embodiments, the surface contains about 1.0×10⁵ to about 1.0×10³⁰ total pores or any number or range of numbers therebetween. In some embodiments, the surface comprises between about 10 and about 1.0×10¹⁵ pores per mm² surface area.

The pores can be distributed in numerous ways within a given surface. In some embodiments, the pores are distributed in parallel within a given surface. In one such example, the pores are distributed side-by-side in the same direction and are the same distance apart within a given surface. In some embodiments, the pore distribution is ordered or homogeneous. In one such example, the pores are distributed in a regular, systematic pattern or are the same distance apart within a given surface. In some embodiments, the pore distribution is random or heterogeneous. In one such example, the pores are distributed in an irregular, disordered pattern or are different distances apart within a given surface. In some embodiments, multiple surfaces are distributed in series. The multiple surfaces can be homogeneous or heterogeneous in surface size, shape, and/or roughness. The multiple surfaces can further contain pores with homogeneous or heterogeneous pore size, shape, and/or number, thereby enabling the simultaneous delivery of a range of compounds into different cell types.

In some embodiments, an individual pore has a uniform width dimension (i.e. constant width along the length of the pore passage). In some embodiments, an individual pore has a variable width (i.e. increasing or decreasing width along the length of the pore passage). In some embodiments, pores within a given surface have the same individual pore depths. In some embodiments, pores within a given surface have different individual pore depths. In some embodiments, the pores are immediately adjacent to each other. In some embodiments, the pores are separated from each other by a distance. In some embodiments, the pores are separated from each other by a distance of about 0.001 μm to about 30 mm or any distance or range of distances therebetween.

In some embodiments, the surface is coated with a material. The material can be selected from any material known in the art, including, without limitation, Teflon, an adhesive coating, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors that modulate cellular function, nucleic acids, lipids, carbohydrates, or transmembrane proteins. In some embodiments, the surface is coated with polyvinylpyrrolidone. In some embodiments, the material is covalently attached to the surface. In some embodiments, the material is non-covalently attached to the surface. In some embodiments, the surface molecules are released at the cells pass through the pores.

In some embodiments, the surface has modified chemical properties. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and/or negatively charged. In some embodiments, the surface can be positively charged in some regions and negatively charged in other regions. In some embodiments, the surface has an overall positive or overall negative charge. In some embodiments, the surface can be any one of smooth, electropolished, rough, or plasma treated. In some embodiments, the surface comprises a zwitterion or dipolar compound.

In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

VI. COMPOUNDS TO DELIVER

In certain aspects, the present disclosure relates to methods for delivering a compound into a cell comprising a cell wall or modified to remove all or part of a cell wall. In some embodiments, the compound is a single compound. In some embodiments, the compound is a mixture of compounds. In some embodiments, a compound or mixture of compounds is delivered to a cell comprising a cell wall or modified to remove all or part of a cell wall to produce a desired effect.

In some embodiments, the compound comprises a nucleic acid. In some embodiments, the compound is a nucleic acid. Exemplary nucleic acids include, without limitation, recombinant nucleic acids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, shRNA, self-amplifying mRNA, and peptide nucleic acids. In some embodiments, the nucleic acid is homologous to a nucleic acid in the cell. In some embodiments, the nucleic acid is heterologous to a nucleic acid in the cell. In some embodiments, the nucleic acid comprises a transposon, and optionally a sequence encoding a transposase. In some embodiments, the compound is a plasmid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid encodes a therapeutic polypeptide. In some embodiments the nucleic acid encodes a reporter or a selectable marker. Exemplary reporter markers include, without limitation, green fluorescent protein (GFP), red fluorescent protein (RFP), auquorin, beta-galactosidase, Uroporphyrinogen (urogen) III methyltransferase (UMT), and luciferase. Exemplary selectable markers include, without limitation, Blasticidin, G418/GENETICIN™ (gentamicin), Hygromycin B, Puromycin, ZEOCIN® (phleomycin), nourseothricin (NTC), Adenine Phosphoribosyltransferase, and thymidine kinase. In some embodiments, the nucleic acid encodes a growth factor or inducer.

In some embodiments, the compound comprises an organelle comprising a nucleic acid. In some embodiments, the compound is an organelle comprising a nucleic acid. In some embodiments, the organelle is a double-membrane organelle, such as a plastid. In some embodiments, the organelle is a plant organelle, such as a plant plastid. In some embodiments, the plant organelle is a plastid that can include, without limitation, chloroplasts, chromoplasts, gerontoplasts, leucoplasts, amyloplasts, elaioplasts, proteinoplasts, and tannosomes. In some embodiments, the organelle is an algal organelle, such as an algal plastid. In some embodiments, the algal organelle is a plastid that can include, without limitation, leucoplasts, muroplasts, and rhodoplasts.

In some embodiments, the compound comprises a protein-nucleic acid complex. In some embodiments, the compound is a protein-nucleic acid complex. In some embodiments, protein-nucleic acid complexes, such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, are used in genome editing applications. These complexes contain sequence-specific DNA-binding domains in combination with nonspecific DNA cleavage nucleases. These complexes enable targeted genome editing, including adding, disrupting, or changing the sequence of a specific gene. In some embodiments, a disabled CRISPR is used to block or induce transcription of a target gene. In some embodiments, the compound contains a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the compound includes a nucleic acid encoding for a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the compound includes a transposase protein and a nucleic acid comprising a transposon.

In some embodiments, the compound comprises a lipid-nucleic acid complex. In some embodiments, the compound is a lipid-nucleic acid complex. In some embodiments, lipid-nucleic acid complexes are used to increase efficiency of delivering the nucleic acid into the cell. Exemplary nucleic acids include any of the nucleic acids described herein. In some embodiments, the lipid-nucleic acid complex comprises a cationic lipid and optionally a neutral helper lipid. In some embodiments, the cationic lipid can include, without limitation, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), [1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP), 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioctadecylamidoglycylspermine (DOGS), and 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). In some embodiments, the neutral helper lipid can include, without limitation, dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). In some embodiments, a lipid in the lipid-nucleic acid complex is modified with a polymer, such as polyethylene glycol (PEG).

In some embodiments, the compound comprises a cation-nucleic acid complex. In some embodiments, the compound is a cation-nucleic acid complex. In some embodiments, cation-nucleic acid complexes are used to increase efficiency of delivering the nucleic acid into the cell. Exemplary nucleic acids include any of the nucleic acids described herein. In some embodiments, the cation is a polycation. In some embodiments, the cation-nucleic acid complex comprises a cation that can include, without limitation, lithium, cesium, calcium, and polylysine (PLL).

In some embodiments, the compound comprises a protein or polypeptide. In some embodiments, the compound is a protein or polypeptide. In some embodiments, the protein or polypeptide is a therapeutic protein, antibody, growth factor or inducer, fusion protein, antigen, synthetic protein, reporter marker, or selectable marker. In some embodiments, the protein is a gene-editing protein such as a zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), mega nuclease, CRE recombinase, FLP recombinase, R recombinase, integrase, or transposase. In some embodiments, the fusion proteins can include, without limitation, chimeric protein drugs such as antibody drug conjugates or recombinant fusion proteins such as proteins tagged with GST or streptavidin. In some embodiments, the compound is a transcription factor. In some embodiments, the compound is chromatin remodeling protein that can include, without limitation, histone acetyltransferases (HATs), deacetylases, methyltransferases, demethylases, and kinases. HATs can include, without limitation, Gcn5, PCAF, Hat1, Elp3, Hpa2, Hpa3, ATF-2, Nut1, MOZ, Ybf2 (Sas3), Sas2, Tip60, Esa1, MOF, MORF, HBO1, p300, CBP, SRC-1, SRC-3, ACTR, TIF-2, TAF_(II)250, TFIIIC, Rtt109, and CLOCK. Deactylases can include, without limitation, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, and Sir2. Methyltransferases can include, without limitation, ASH1L, DOT1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, PRDM2, SET, SETBP1, SETD1A, SETD1B, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETD9, SETDB1, SETDB2, SETMAR, SMYD1, SMYD2, SMYD3, SMYD4, SMYD5, SUV39H1, SUV39H2, SUV420H1, and SUV420H2. Demethylases can include, without limitation, KDM1A, KDM1B, KDM2A, KDM2B, KDM3A, KDM3B, JMJD1C, KDM4A, KDM4B, KDM4C, KDM4D, KDM5A, KDM5B, KDM5C, KDM5D, KDM6A, KDM6B, and UTY.

In some embodiments the protein or polypeptide is a reporter or a selectable marker. Exemplary reporter markers include, without limitation, green fluorescent protein (GFP), red fluorescent protein (RFP), auquorin, beta-galactosidase, Uroporphyrinogen (urogen) III methyltransferase (UMT), and luciferase. Exemplary selectable markers include, without limitation, Blasticidin, G418/GENETICIN™ (gentamicin), Hygromycin B, Puromycin, ZEOCIN® (phleomycin), Adenine Phosphoribosyltransferase, and thymidine kinase.

In some embodiments, the compound comprises an antibody. In some embodiments, the compound is an antibody. In some embodiments, the antibody is a full length antibody or an antibody fragment. Antibodies for use in the present disclosure include, without limitation, antibody variants, labeled antibodies, antibody fragments such as Fab or F(ab)₂ fragments, single-domain antibodies, single-chain antibodies, multi-specific antibodies, antibody fusion proteins, and immunoadhesins. The antibodies may be any isotype known in the art, including IgA, IgG, IgE, IgD, or IgM.

In some embodiments, the compound comprises a small molecule. In some embodiments, the compound is a small molecule. Exemplary small molecules include, without limitation, fluorescent markers, dyes, pharmaceutical agents, metabolities, or radionucleotides. In some embodiments, the pharmaceutical agent is a therapeutic drug and/or cytotoxic agent.

In some embodiments, the compound comprises a nanoparticle. Examples of nanoparticles include gold nanoparticles, quantum dots, carbon nanotubes, nanoshells, dendrimers, and liposomes. In some embodiments, the nanoparticle contains a therapeutic molecule. In some embodiments, the nanoparticle contains a nucleic acid, such as mRNA. In some embodiments, the nanoparticle contains a label, such as a fluorescent or radioactive label.

In some embodiments, the compound comprises a fluorescently tagged molecule. In some embodiments, the compound is a fluorescently tagged molecule, such as a molecule tagged with a fluorochrome such as pacific blue, Alexa 288, Cy5, or cascade blue. In some embodiments, the compound is a radionucleotide, dextran particle, magnetic bead, or impermeable dye. In some embodiments, the compound is a 3 kDa dextran particle labeled with PacBlue. In some embodiments, the compound is a 10 kDa dextran particles labeled with Alexa488. In some embodiments, the compound is a small molecule fluorophore tagged protein. In some embodiments, the compound is a small molecule tagged with Alexa647.

In some embodiments, the compound to deliver is purified. In some embodiments, the compound is at least about 20% by weight (dry weight) the compound of interest. In some embodiments, the purified compound is at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% the compound of interest. In some embodiments, the purified compound is at least about 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) the compound of interest. Purity is determined by any known methods, including, without limitation, column chromatography, thin layer chromatography, HPLC analysis, NMR, mass spectrometry, or SDS-PAGE. Purified DNA or RNA is defined as DNA or RNA that is free of exogenous nucleic acids, carbohydrates, and lipids.

VII. CELL PERTURBATIONS

In certain aspects, the present disclosure relates to passing a cell suspension through a constriction, wherein the constriction deforms the cell comprising a cell wall or modified to remove (such as digest or disrupt) all or part of a cell wall, causing a perturbation of the cell. In some embodiments, the perturbation is a perturbation in the cell wall. In some embodiments, the perturbation is a perturbation in the cell membrane. In some embodiments, the perturbation is a perturbation in both the cell wall and the cell membrane. In some embodiments, the cell wall is treated prior to passing through the constriction to alter the cell wall. In some embodiments, the cell wall is treated using chemicals, enzymes such as cellulase, pectinase, xylanase, lysozyme, zymolyase, or chitinase, heat, or ultrasound prior to passing through the constriction. In some embodiments, treating a cell with an enzyme such as cellulase, pectinase, xylanase, lysozyme, zymolyase, or chitinase results in digestion of all or part of the cell wall. In some embodiments, treating a cell with heat or ultrasound results in disruption of all or part of the cell wall. In some embodiments, the cell wall is treated more than about any of 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours or any time therebetween prior to passing the cell through the constriction. In some embodiments, disruption of the well is disruption of more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cell wall. Methods to measure cell wall disruption are known in the art; for example, confocal microscopy, electron microscopy, atomic force microscopy (Dupre), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In some embodiments, the cell wall is not treated prior to passing through the constriction. The deformation in the cell can be caused by, for example, pressure induced by mechanical strain and/or shear forces. The perturbation in the cell is a breach in the cell that allows material from outside the cell to move into the cell (e.g., a hole, tear, cavity, aperture, pore, break, gap, perforation). In some embodiments, the perturbation is transient. In some embodiments, as the cell passes through the constriction, the deformation temporarily imparts injury to the cell wall that causes passive diffusion of material through the perturbation. In some embodiments, the constriction deforms the cell to about any one of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the diameter of the cell or any value therebetween. In some embodiments, the passage of the compound into the cell occurs simultaneously with the cell passing through the constriction and/or the perturbation of the cell. In some embodiments, passage of the compound into the cell occurs after the cell passes through the constriction.

VIII. DELIVERY PARAMETERS

In certain aspects, the present disclosure relates to methods for delivering a compound into a cell comprising a cell wall including the steps of passing a cell suspension through a constriction, wherein the constriction deforms the cell comprising a cell wall, causing a perturbation of the cell, and contacting the cell suspension with the compound. In other aspects, the present disclosure relates to methods for delivering a compound into a cell modified to remove all or part of a cell wall including the steps of passing a cell suspension through a constriction, wherein the constriction deforms the cell modified to remove all or part of a cell wall, causing a perturbation of the cell, and contacting the cell suspension with the compound. The cell suspension may be contacted with the compound before, concurrently, or after passing through the constriction. The cell comprising a cell wall or modified to remove all or part of a cell wall may pass through the constriction suspended in a solution that includes the compound to deliver, although the compound can be added to the cell suspension after the cells pass through the constriction. In some embodiments, the compound to be delivered is coated on the constriction.

Several parameters can influence the delivery of the compound into the cell comprising a cell wall or modified to remove all or part of a cell wall. For example, the dimensions of the constriction, the entrance angle of the constriction, the surface properties of the constrictions (e.g. roughness, chemical modification, hydrophilic, hydrophobic, etc.), the operating flow speeds (e.g., cell transit time to the constriction), the cell concentration, the concentration of the compound in the cell suspension, and the amount of time that the cell recovers or incubates after passing through the constrictions can affect the passage of the delivered compound into the cell. Additional parameters influencing the delivery of the compound into the cell can include the velocity of the cell in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component that is perpendicular to flow velocity, and time in the constriction. Such parameters can be designed to control delivery of the compound. In some embodiments, the cell concentration ranges from about 10 to about 10²⁰ cells/ml or any concentration or range of concentrations therebetween. In some embodiments, the cell concentration ranges from about 10 to about 10², 10² to about 10³, 10³ to about 10⁴, 10⁴ to about 10⁵, 10⁵ to about 10⁶, 10⁶ to about 10⁷, 10⁷ to about 10⁸, 10⁸ to about 10⁹, 10⁹ to about 10¹⁰, 10¹⁰ to about 10¹¹, 10¹¹ to about 10¹², 10¹² to about 10¹³, 10¹³ to about 10¹⁴, 10¹⁴ to about 10¹⁵, 10¹⁵ to about 10¹⁶, 10¹⁶ to about 10¹⁷, 10¹⁷ to about 10¹⁸, 10¹⁸ to about 10¹⁹, 10¹⁹ to about 10²⁰, or any concentration or range of concentrations therebetween. In some embodiments, delivery compound concentrations can range from about 10 ng/ml to about 1 g/mL or any concentration or range of concentrations therebetween. In some embodiments, delivery compound concentrations can range from about 1 pM to about 2M or any concentration or range of concentrations therebetween.

The temperature used in the methods of the present disclosure can be adjusted to affect compound delivery and cell viability. In some embodiments, the method is performed between about −5° C. and about 45° C. For example, the methods can be carried out at room temperature (e.g., about 20° C.), physiological temperature (e.g., about 37° C.), higher than physiological temperature (e.g., greater than about 37° C. to 45° C. or more), or reduced temperature (e.g., about −5° C. to about 4° C.), or temperatures between these exemplary temperatures.

Various methods can be utilized to drive the cells comprising a cell wall through the constrictions. For example, pressure can be applied by a pump on the entrance side (e.g., gas cylinder, or compressor), a vacuum can be applied by a vacuum pump on the exit side, capillary action can be applied through a tube, and/or the system can be gravity fed. Displacement based flow systems can also be used (e.g., syringe pump, peristaltic pump, manual syringe or pipette, pistons, etc.). In some embodiments, the cells are passed through the constrictions by positive pressure or negative pressure. In some embodiments, the cells are passed through the constrictions by constant pressure or variable pressure. In some embodiments, pressure is applied using a syringe. In some embodiments, pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump. In some embodiments, pressure is applied using a vacuum. In some embodiments, the cells are passed through the constrictions by g-force. In some embodiments, the cells are passed through the constrictions by capillary pressure.

In some embodiments, fluid flow directs the cells through the constrictions. In some embodiments, the fluid flow is turbulent flow prior to the cells passing through the constriction. Turbulent flow is a fluid flow in which the velocity at a given point varies erratically in magnitude and direction. In some embodiments, the fluid flow through the constriction is laminar flow. Laminar flow involves uninterrupted flow in a fluid near a solid boundary in which the direction of flow at every point remains constant. In some embodiments, the fluid flow is turbulent flow after the cells pass through the constriction.

The velocity at which the cells pass through the constrictions can be varied. In some embodiments, the cells pass through the constrictions at a uniform cell speed. In some embodiments, the cells pass through the constrictions at a fluctuating cell speed.

IX. EXEMPLARY EMBODIMENTS

A method for delivering a compound into a cell comprising a cell wall, the method comprising passing a cell suspension through a constriction, wherein said constriction deforms the cell comprising a cell wall, thereby causing a perturbation of the cell such that the compound enters the cell, wherein said cell suspension is contacted with the compound.

A method for delivering a compound into a cell modified to remove all or part of a cell wall, the method comprising passing a cell suspension through a constriction, wherein said constriction deforms the cell modified to remove all or part of a cell wall, thereby causing a perturbation of the cell such that the compound enters the cell, wherein said cell suspension is contacted with the compound.

A method for delivering a compound into a cell comprising a cell wall, the method comprising a) removing all or part of the cell wall, and b) passing the cell suspension through a constriction, wherein said constriction deforms the cell modified to remove all or part of a cell wall, thereby causing a perturbation of the cell such that the compound enters the cell, wherein said cell suspension is contacted with the compound.

The method of any one of embodiments 2-3, wherein all or part of the cell wall is removed prior to passing the cell suspension through the constriction by treating the cell suspension with an enzyme, heat or ultrasound.

The method of embodiment 4, wherein all or part of the cell wall is removed by treatment with an enzyme.

The method of embodiment 5, wherein the enzyme is one or more of cellulase, pectinase, xylanase, lysozyme, zymolyase, or chitinase.

The method of any one of embodiments 1-6, wherein the constriction is contained within a microfluidic channel.

The method of any one of embodiments 1-7, wherein the constriction is a pore or contained within a pore.

The method of embodiment 8, wherein the pore is contained in a surface.

The method of embodiment 9, wherein the surface is a filter.

The method of embodiment 9, wherein the surface is a membrane.

The method of any one of embodiments 1-11, wherein the constriction size is a function of the cell diameter.

The method of any one of embodiment 1-12, wherein the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.

The method of any one of embodiments 1-13, wherein the cell suspension comprises a mixed cell population.

The method of any one of embodiments 1-13, wherein the cell suspension comprises a purified cell population.

The method of any one of embodiments 1-15, wherein the cell comprising a cell wall or modified to remove all or part of a cell wall is a plant, yeast, fungal, algal, or prokaryotic cell.

The method of embodiment 16, wherein the plant cell is a crop, model, ornamental, vegetable, leguminous, conifer, or grass plant cell.

The method of embodiment 16, wherein the yeast cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain cell.

The method of embodiment 16, wherein the fungal cell is an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma strain cell.

The method of embodiment 16, wherein the prokaryotic cell is a Bacillus coagulans, Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Carboxydocella sp., Clostridium perfringens, Clostridium septicum, Clostridium tetani, Corynebacterium glutamicum, Enterobacteriaceae, Enterococcus faecalis, Erwinia chrysanthemi, Faecalibacterium prausnitzii, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sp., Pediococcus acidilactici, Peptococcus sp., Peptostreptococcus sp., Propionibacterium freudenreichii, Proteus mirabilis, Pseudomonas aeruginosa, Rhodopseudomonas capsulata, Salmonella enteritidis, Staphylococcus aureus, Streptococcus faecium, Streptococcus lactis, Streptococcus salivarius, Streptococcus thermophilus, Vibrio furnissii, Caldicellulosiruptor saccharolyticus, Xanthomonas campestris, cyanobacteria, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, or Paracoccus cell.

The method of embodiment 16, wherein the algal cell is a Chlorophyceae, Bacillariophyceae, Eustigmatophyceae, or Chrysophyceae cell.

The method of any one of embodiments 1-21, wherein the compound comprises a nucleic acid.

The method of any one of embodiments 1-22, wherein the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplifying mRNA.

The method of any one of embodiments 1-23, wherein the compound comprises a transposon.

The method of any one of embodiments 1-24, wherein the compound is a plasmid.

The method of any one of embodiments 1-23, wherein the compound comprises a plastid.

The method of any one of embodiments 1-23, wherein the compound comprises a polypeptide-nucleic acid complex.

The method of any one of embodiments 1-23 or 27, wherein the compound comprises a Cas9 protein and a guide RNA or donor DNA.

The method of any one of embodiments 1-23, wherein the compound comprises nucleic acid encoding for a Cas9 protein and a guide RNA or donor DNA.

The method of any one of embodiments 1-23 or 27, wherein the compound comprises a transposase protein and a nucleic acid comprising a transposon.

The method of any one of embodiments 1-25, wherein the compound comprises a lipid-nucleic acid complex.

The method of any one of embodiments 1-25, wherein the compound comprises a cation-nucleic acid complex.

The method of any one of embodiments 1-21, wherein the compound comprises a polypeptide or peptide.

The method of any one of embodiments 1-22 or 33, wherein the compound comprises a TALEN protein, Zinc finger nuclease, mega nuclease, CRE recombinase, FLP recombinase, R recombinase, integrase, or transposase.

The method of any one of embodiments 1-21 or 33, wherein the compound comprises a histone acetyltransferase, deacetylase, methyltransferase, or demethylase.

The method of any one of embodiments 1-21 or 33, wherein the compound is an antibody.

The method of any one of embodiments 1-21 or 33, wherein the compound is a transcription factor.

The method of any one of embodiments 1-21, wherein the compound is a small molecule.

The method of any one of embodiments 1-21, wherein the compound is a nanoparticle.

The method of any one of embodiments 1-21, wherein the compound is a liposome.

The method of any one of embodiments 1-21, wherein the compound is a fluorescently tagged molecule.

Examples Example 1: Delivery to S. cerevisiae Cells

In order to evaluate the constriction-mediated delivery of molecules into cells comprising cell walls, S. cerevisiae cells mixed with fluorescent dextran particles are passed through a constriction, and intracellular particle delivery is evaluated. The cells are pre-incubated with chemicals to remove the cell wall prior to passing through the constriction. Alternatively, the cells are not incubated with chemicals and the cell wall is intact prior to passing through the constriction. The cells are passed through a constriction within a microfluidic channel, or through a surface containing pores. Pressure, temperature, and buffer composition are optimized to achieve delivery. Fluorescence detection is used to measure the delivery efficiency of the dextran particles into the cells.

In vitro studies were carried out to evaluate the constriction-mediated delivery of molecules into S. cerevisiae. S. cerevisiae cells were grown overnight in yeast extract-peptone-dextrose (YPD) medium. Cells were treated with 0.3 U zymolyase for nine minutes at room temperature to partially digest cell walls without significant cell lysis. Treated cells were collected by centrifugation and resuspended at 1×10⁸ cells/mL in PBS at room temperature. The resuspended cells were mixed with 10 μg/mL plasmid DNA for expressing the nourseothricin N-acetyl transferase (NAT) gene, which confers resistance to nourseothricin (NTC), and 11 μM of free Alexa Fluor® 488, and passed through a track etched polycarbonate surface containing 3 μm pores. Additionally, cells mixed with either plasmid DNA alone or plasmid DNA+ free Alexa Fluor® 488, without constriction, were included as endocytosis controls. The cells were then characterized by flow cytometry where dead cells were stained with propidium iodide and excluded from analysis. As shown in FIG. 1 (top row of dot-plots), passage through the pores resulted in fewer live cells compared to the endocytosis controls, as can be expected upon perturbation of the cell membrane. Live cells were then assessed for incorporation of Alexa Fluor® 488 by flow cytometry. Endocytosis accounted for only a small portion of Alexa Fluor® 488-positive cells following constriction, as indicated by the difference in peak shift between the DNA+Alexa Fluor® 488 endocytosis control condition and the constriction conditions shown in FIG. 1 (bottom row of histograms).

Live cells were spread on YPD agar plates containing NTC and incubated for 3 days to allow for growth of S. cerevisiae cells that had successful delivery of the plasmid DNA and expression of the NTC resistance gene. As shown in FIG. 2 , NTC-resistant colonies were present on plates spread with cells mixed with the plasmid DNA and passed through the pores, but not on the plates spread with cells mixed with plasmid DNA without constriction. Individual colonies were re-streaked on selection plates containing NTC, confirming delivery and expression of the plasmid DNA. These results demonstrate that the constriction-mediated delivery approaches described herein are practical for use in cells having a cell wall. 

1. A method for delivering a compound into a cell comprising a cell wall, the method comprising passing a cell suspension which comprises the cell through a constriction, wherein the constriction deforms the cell, thereby causing a perturbation of the cell such that the compound enters the cell through the perturbation when contacted with the cell.
 2. A method for delivering a compound into a cell, which has been modified to remove all or part of a cell wall, the method comprising passing a cell suspension which comprises the cell through a constriction, wherein the constriction deforms the cell, thereby causing a perturbation of the cell such that the compound enters the cell through the perturbation when contacted with the cell.
 3. A method for delivering a compound into a cell comprising a cell wall, the method comprising a) removing all or part of the cell wall, and b) passing a cell suspension which comprises the cell through a constriction, wherein the constriction deforms the cell, thereby causing a perturbation of the cell such that the compound enters the cell through the perturbation when contacted with the cell.
 4. The method of claim 3, wherein the removing all or part of the cell wall comprises treating the cell suspension with an enzyme, heat, or ultrasound.
 5. The method of claim 4, wherein all or part of the cell wall is removed by treatment with an enzyme.
 6. The method of claim 5, wherein the enzyme is one or more of cellulase, pectinase, xylanase, lysozyme, zymolyase, or chitinase
 7. The method of claim 1, wherein the constriction is contained within a microfluidic channel.
 8. The method of claim 1, wherein the constriction is a pore or contained within a pore.
 9. The method of claim 8, wherein the pore is contained in a surface.
 10. The method of claim 9, wherein the surface is a filter or a membrane.
 11. (canceled)
 12. The method of claim 1, wherein the constriction size is a function of the cell diameter.
 13. The method of claim 1, wherein the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.
 14. The method of claim 1, wherein the cell suspension comprises a mixed cell population or a purified cell population.
 15. (canceled)
 16. The method of claim 1, wherein the cell is a plant, yeast, fungal, algal, or prokaryotic cell.
 17. The method of claim 16, wherein: (a) the plant cell is a crop, model, ornamental, vegetable, leguminous, conifer, or grass plant cell; (b) the yeast cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain cell; (c) the fungal cell is an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma strain cell; (d) the prokaryotic cell is a Bacillus coagulans, Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Carboxydocella sp., Clostridium perfringens, Clostridium septicum, Clostridium tetani, Corynebacterium glutamicum, Enterobacteriaceae, Enterococcus faecalis, Erwinia chrysanthemi, Faecalibacterium prausnitzii, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sp., Pediococcus acidilactici, Peptococcus sp., Peptostreptococcus sp., Propionibacterium freudenreichii, Proteus mirabilis, Pseudomonas aeruginosa, Rhodopseudomonas capsulata, Salmonella enteritidis, Staphylococcus aureus, Streptococcus faecium, Streptococcus lactis, Streptococcus salivarius, Streptococcus thermophilus, Vibrio furnissii, Caldicellulosiruptor saccharolyticus, Xanthomonas campestris, cyanobacteria, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, or Paracoccus cell; or (e) the algal cell is a Chlorophyceae, Bacillariophyceae, Eustigmatophyceae, or Chrysophyceae cell. 18-21. (canceled)
 22. The method of claim 1, wherein the compound comprises: (a) a nucleic acid; (b) a transposon; (c) a plasmid; (d) a plastid; (e) a polypeptide-nucleic acid complex; (f) a lipid-nucleic acid complex; (g) a cation-nucleic acid complex; (h) a polypeptide or peptide; (i) a small molecule; (j) a nanoparticle; (k) a liposome; or (l) a fluorescently tagged molecule.
 23. The method of claim 22, wherein the nucleic acid comprises a DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplifying mRNA. 24-27. (canceled)
 28. The method of claim 1, wherein the compound comprises: (a) a Cas9 protein and a guide RNA or donor DNA; (b) a nucleic acid encoding for a Cas9 protein and a guide RNA or donor DNA; (c) a transposase protein and a nucleic acid comprising a transposon; (d) a TALEN protein, Zinc finger nuclease, mega nuclease, CRE recombinase, FLP recombinase, R recombinase, integrase, or transposase; (e) a histone acetyltransferase, deacetylase, methyltransferase, or demethylase; (f) an antibody; or (g) a transcription factor. 29-41. (canceled) 